ABSTRACT Translation is the cellular process that converts DNA into proteins, and defects in this process are linked to many human diseases. A key component in translation is transfer RNA (tRNA), which must be extensively chemically modified by numerous cellular enzymes to function properly. Many of these enzymes and the tRNA modifications that they form are conserved in eukaryotic organisms ranging from single-celled budding yeast to multi-cellular organisms such as humans, thus making yeast a powerful model organism for studying the roles of modifications in the cell. However, some modifications found on human tRNAs are not found in yeast, and much less is known about human tRNA modifications in general. Because defects in tRNA modifications are strongly linked to several human diseases, this NIH R15 AREA proposal seeks to expose undergraduate students to modern biomedical research by advancing the field of human tRNA modification research in three ways. First, we propose to study the yeast and human proteins required to form two related, conserved tRNA modifications. Defects in these modifications cause intellectual disability and are linked to type 2 diabetes and polycystic ovary syndrome. Thus, in Aim 1 of the proposal, students will identify regions in the tRNA modifying proteins Trm7, Trm732, and Trm734 required for function and intramolecular interactions. These proteins are required for the addition of a 2?-O-methyl group on positions 32 and 34 in the critical anticodon loop region of yeast tRNAs. Students will use bioinformatics along with genetic, molecular biological, and biochemical approaches to identify these regions. Second, we propose to determine if the corresponding human Trm7, Trm732, and Trm734 proteins are required for methylation of 32 and 34 on human and animal tRNA. This research will follow up on our intriguing preliminary data suggesting that these methylations are formed by two different mechanisms on human tRNAs. Thus, in Aim 2 of our proposal, students will quantify the levels of these modifications on tRNAs purified from human and fruit fly cell lines lacking the predicted Trm7, Trm732, and Trm734 genes. Third, we propose to identify the gene required to form a human tRNA anticodon loop modification that is not found in yeast. Thus, in Aim 3 we propose to identify the enzyme that adds a 2?-O- methyl group to position 39 of certain human and animal tRNAs. Students will use bioinformatics to identify candidate genes that could be required for this modification, and then determine if the genes are responsible using a rapid gene knockdown technique in cultured fruit fly cells. Once identified in fruit flies, we will determine if the corresponding human gene is required for the modification activity on human tRNA. The research proposed herein meets the criteria of the NIH R15 AREA award because it gives undergraduate students the opportunity to participate in a holistic, modern research approach that will increase knowledge of the underlying molecular causes of disease. Undergraduates will be trained in genetic, molecular biological, biochemical, and bioinformatics techniques, helping prepare a new generation of biomedical researchers.